Staphylococcus epidermidis causes about 250,000 hospital-acquired infections per year in the U.S.A., making it the leading cause of nosocomial infections. Although the type and severity of diseases produced by this opportunistic pathogen varies, its most common infectious manifestation is associated with indwelling medical devices (e.g., catheters). Catheter associated infections usually involve the formation of a bacterial biofilm, a process requiring the production of polysaccharide intercellular adhesin (PIA). PIA is a ?-1, 6-linked polysaccharide that is strongly associated with the staphylococcal cell surface and mediates cell-to-cell adhesion. Synthesis of PIA increases during growth in either oxygen limiting, nutrient replete, or iron-limiting conditions. Importantly, these same environmental conditions alter the metabolic status of the bacteria, thus, creating an effective means to convey extracellular environmental changes to the intracellular environment. The long-term goal is to understand how staphylococci regulate virulence factor synthesis in response to environmental stimuli. The objective of this application is to determine how extracellular environmental factors alter the intracellular metabolic status to affect changes in S. epidermidis PIA synthesis. The central hypothesis of this application is that the environmental factors regulating PIA synthesis do so through the inhibition of a common metabolic pathway, specifically, the tricarboxylic acid (TCA) cycle. The genesis of this hypothesis was the observation that the environmental factors influencing PIA production also affect TCA cycle function. Strong support for this hypothesis is based on the observation that incubation of S. epidermidis with low concentrations of a TCA cycle inhibitor dramatically increases PIA production. The central hypothesis predicts that inducing TCA cycle activity will reduce or eliminate PIA production. Therefore, blocking the transport of a key TCA cycle metabolite or amino acid will require an active TCA cycle for bacterial survival, limiting PIA synthesis and reducing the ability S. epidermidis to form a biofilm. The research contained within this proposal will have a catalytic impact in determining which membrane transporters to target for vaccine development. It is anticipated that vaccines directed against membrane transporters of key TCA cycle molecules, or amino acids, will prevent the maturation of a S. epidermidis biofilm, thus, enhancing the likelihood of successfully treating the infection. [unreadable] [unreadable] [Lay summary] Many bacterial infections are difficult for physicians treat because the bacteria can grow in dense clusters known as biofilms. In order for bacteria to form a biofilm, they must produce a complex sugar to hold the biofilm together. The work contained in this proposal is a first step toward inhibiting the formation of this complex sugar, preventing the formation of a bacterial biofilm, and providing physicians a way to treat patients. [unreadable] [unreadable]